Method for detecting chitin-containing organisms

ABSTRACT

The present invention provides a method for detecting chitin-containing organisms on an area of a person or animal by contacting the particular area with a dye that is capable of binding or conjugating to chitin and emitting fluorescence upon exposure to light. If a chitin-containing organism is present in the treated area, the chitin of the organism will bind or conjugate the dye and, upon exposure to light, the chitin-containing organisms may be visualized and removed. Also provided by the present invention are solutions and suspensions that contain a dye capable of binding or conjugating to a chitin-containing organism and emitting fluorescence upon exposure to light. The solutions and suspensions provided herein may be in the form of a shampoo, cream, lotion or detergent for the detection of chitin-containing organisms present in body hair, on the skin, clothing or the fur of animals. The solutions and suspensions may also be in a form suitable for spraying on clothes, as well as on the hair, skin and fur. Further provided by the present invention are kits comprising a solution or suspension that contains a dye capable of binding or conjugating to a chitin-containing organism and a source of light that emits a wavelength capable of exciting fluorescence from the chitin-bound or chitin-conjugated dye.

BACKGROUND OF THE INVENTION

[0001] Numerous human and animal diseases are caused by the bite of orinfestation by arthropods. Arthropods include insects such as lice andarachnids such as mites and ticks. Because of the small size of thesecreatures and their larvae, it is often difficult to detect theirpresence on people or their pets.

[0002] Human pediculosis, or lice, is caused by infestation of the head,body, or pubic area by the arthropods Pediculus capitus, Pediculushumanus, Pediculus corporis or Phithirus pubis, respectively. Head licelay eggs, called nits or louse eggs, on the hair of the head, and thenits eventually hatch into mature forms. Body lice lay their eggs inwarm moist skin crease areas. Treatment is with shampoos, creams andlotions containing various insecticides such as lindane, Malathion, orpermethrin. Following treatment, the residual nits must be removed bycareful examination of the affected hair or clothing and mechanicalremoval. The shampoo treatment does not remove nits which tenaciouslyadhere to hair.

[0003] Most pre-schools and elementary schools in the U.S. have periodicchecks of their students for lice to limit infections. The child's headis checked for the presence of either nits or lice, and if found, thechild is sent home for treatment. After treatment, the child isrechecked in school. Often, however, a successfully treated child willhave residual nits that were missed and not removed. This usuallyresults from an inability to see the small (0.8 mm×0.3 mm) eggs thatblend in well with hair, especially light hair. For the untrainedobserver, it is often difficult to differentiate nits from exfoliatedscalp skin (dandruff). This may lead to further embarrassment of a childwho might have been treated but is sent home again after findingresidual nits.

[0004] U.S. Pat. No. 4,830,790, issued Apr. 19, 1993 to Saferstein, A.for “Multi-Function Light Device” describes a source of blacklight bluelight for the detection of nits and adult lice. Since autofluorescenceof nits and adult lice is minimal, however, it is still extremelydifficult to detect nits and adult lice in hair and clothing.

[0005] Lyme disease or erythema chronicum migrans is a multi systemdisease affecting the skin joints and central nervous system caused bythe organism Borrelia burgdorefi. The disease is contracted by the biteof the deer tick Ixodes scapularis which secretes the Borrelia organismin its saliva when it feeds off the human host. Other tick vectors foundto be associated with the Borrelia organism are Ixodes pacificus, foundin the west, I. ricinus, found in Europe, and I. persulcatus, found inAsia. Rocky Mountain spotted fever is an acute rickettsial disease thatis transmitted by the bite of a wood tick, Dermacentor andersoni. Rapidremoval of the deer and wood ticks after attachment could prevent thetransmission of the Borrelia and Dermacentor organisms and thus preventLyme disease or Rocky Mountain spotted fever, but is often difficultsince the attachment and bite of the tick are most often not seen orfelt by the host.

[0006] Other organisms that are small in size and thus difficult todetect are mites, such as chiggers. These organisms also cause humandisease. Accordingly, a need remains for an effective and expedientmethod of detecting small organisms, such as lice, nits, mites, ticks,and fungi.

SUMMARY OF THE INVENTION

[0007] The present invention provides a precise and efficient method ofdetecting chitin-containing organisms such as lice, nits, mites, ticks,and fungal infections of the skin. The method provided herein enables aquick and accurate detection of chitin-containing organisms on areassuch as the hair, skin and clothing of humans and the fur and skin ofanimals.

[0008] The method provided by the present invention involves the stepsof treating the hair, scalp, skin, or clothing with a dye thatfluoresces when exposed to ultraviolet or visible light. If achitin-containing organism is present in the treated area, the chitin ofthe organism will bind the dye. The area is then exposed to ultravioletlight and the chitin-containing organism fluoresces and can bevisualized and removed.

[0009] The present invention also provides solutions and suspensionsthat contain a dye capable of binding to a chitin-containing organism inan amount effective to bind to a chitin-containing organism and emitfluorescence upon exposure to light. The solutions and suspensionsprovided herein may be in the form of a shampoo, cream, lotion ordetergent for the detection of chitin-containing organisms present inbody hair, on the skin, clothing or the fur of animals. The solutionsand suspensions may also be in a form suitable for spraying on clothes,as well as on the hair, skin and fur.

[0010] The present invention also provides a kit comprising a solutionor suspension that contains a dye capable of binding to achitin-containing organism and a source of light that emits a wavelengthcapable of exciting fluorescence from the chitin-bound orchitin-conjugated dye.

[0011] The method provided herein is extremely useful for the detectionof nits, which are difficult to see with the naked eye. Another usefulapplication of the novel method of the present invention is thedetection of ticks, such as the deer tick which carries Borrelia, theorganism which causes Lyme disease. Deer ticks are extremely small, andmust be immediately removed from the site of attachment in order toprevent transmission of the Borrelia organism.

[0012] Additional objects of the invention will be apparent from thedescription which follows.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a precise and efficient method ofdetection of chitin-containing organisms. The method provided by thepresent invention allows for the visualization of chitin-containingorganisms by treating an area of a human or animal with a dye thatrenders the chitin-containing organism fluorescent when exposed toultraviolet or visible light. Chitin is a nitrogen containingpolysaccharide within a class of polymers of N-acetylglucosamine presentin the covering layer of insects and in the cell walls of many fungi. Inparticular, chitin is a major component of arthropod shells and the cellwalls of lice, nits, mites, ticks, and fungi. Chitin is suitable forstaining and is unique to the arthropod while absent from humans andother mammals.

[0014] The present invention provides a method for detectingchitin-containing organisms. The novel method provided herein employs adye that stains chitin by binding or conjugating to chitin andfluoresces when exposed to ultraviolet or visible light. The particulardye is capable of fluorescing only when bound or conjugated to chitin.The dye is placed in contact with the area to be tested, and thechitin-containing organisms that come into contact with the dye willabsorb the dye and fluoresce upon exposure to ultraviolet or visiblelight. The organism can then be easily located and removed.

[0015] The preferred dyes employed in the method of the presentinvention are those that both bind or conjugate chitin and fluoresce inultraviolet or visible light. Non-limiting examples of dyes that bind orconjugate chitin and fluoresce in ultraviolet or visible light includecalcofluor white, Uvitex 3B, Rylux BA, Rylux BSU (Ostacolor, Pardubice,Czech Republic) and Blankophor (Bayer). Calcofluor white and Uvitex 3Bare colorless dyes that fluoresce brightly on exposure to long waveultraviolet light and short wavelength visible light. Calcofluor whitemay be obtained as fluorescent brightener 28 (Tinopal UNPA-GX) in thesalt or the free acid form (Sigma Chemical Co., St. Louis, Mo.) orcalcofluor white M2R (Poly-sciences, Inc., Warrington, Pa., or SigmaChemical Co., St. Louis, Mo.). A preferred concentration of calcofluorwhite or Uvitex B for use in the direct application to the skin of ahuman or animal is 1% of the dye dissolved in distilled water, althougheffective concentrations may be anywhere between 0.01%-5%, 0.01-1.5%,0.005-2&, 0.75-1.3% or 0.01-10%.

[0016] Examples of arthropods that can be detected using the methodprovided herein include lice, ticks, and mites, such as chiggers.Examples of lice that may be detected include head lice, body lice, andpubic or crab lice. Head lice, caused by the infestation of Pediculuscapitus, is the most common variety to appear on the head and scalp.Body lice, cause by the infestation of Pediculus humanus, are usuallyfound on clothing. Phithirus pubis usually infests pubic hair, but canalso be found on the eyelashes, eyebrows, mustache, or beard. Alsodetectable by the method of the present invention are nits, or louseeggs. Head lice and pubic lice most commonly attach their eggs to hair,while body lice attach their eggs to clothing fibers. These areas can betreated using the method of the present invention to locate and removenits.

[0017] The method of the present invention also detects fungalinfections caused by organisms such as blastomyces, aspergillus,cryptococcus and candida. Also detectable by the method described hereinare infections caused by dermatophytic fungi that manifest as ringworm.Examples of fungal infections that are detectable using the novel methodof the present invention are tinea capitis, a fungal infection of thescalp, caused by Microsporum and Trichophyton species; tinea corporis, afungal infection of the body, caused by Epidermophyton, Microsporum andTrichophyton species; tinea pedis, otherwise known as athlete's foot,caused by Epidermophyton and Trichophyton species; tinea unguium, afungal infection of the nails, caused by Trichophyton species; and tineacruris, a fungal infection of the groin, caused by Trichophyton andEpidermophyton species. Also detectable by the method of the presentinvention is the ringworm caused by Microsporum canis, a fungus whichcommonly infects dogs and cats.

[0018] The present invention further provides a solution or suspensioncomprising a dye capable of binding or conjugating to achitin-containing organism in an amount effective to bind or conjugateto a chitin-containing organism and emit fluorescence upon exposure tolight. The solution or suspension provided herein may contain, forexample, a dye such as calcofluor white or Uvitex 3B. The solution orsuspension may comprise about 0.01% to 10% of the dye, with thepreferred amount being about 1%. The solution or suspension of thepresent invention may be in the form of a shampoo, cream, lotion,detergent, or spray. In a preferred embodiment of the invention, the dyeis incorporated into a shampoo and nits are visualized with ultravioletlight after shampooing. In another embodiment, the dye is incorporatedin a cream or lotion formulation. In a further embodiment of theinvention, the colorless dye that fluoresces upon exposure toultraviolet light is contained in a solution or suspension which issuitable for spraying on hair, skin, clothing, and the fur of animalsfor the visualization of organisms upon exposure to ultraviolet light.Since the colorless dye fluoresces only when bound or conjugated tochitin, the solution does not need to be removed from the surroundingarea to visualize the fluorescing tick. The fluorescent ticks, mites,nits, or other organism can then be pinpointed and removed. Thisembodiment of the invention is extremely useful for visualizingorganisms that are difficult to detect because of their small size, suchas ticks, lice, and mites. This embodiment of the invention isparticularly useful for the visualization of the deer tick, Ixodesscapularis, which carries Borrelia burgdorefi, the causative agent ofLyme disease. A further, very practical use of this embodiment is thevisualization of nits for rapid and accurate detection. For example, thespray solution or suspension can be used to quickly detect the presenceof lice and their eggs on hair and clothing as part of screeningprograms in schools, camps, physician's offices, pediatric clinics andemergency rooms. The spray solution or suspension may also be used todetect chitin-containing organisms on furniture, bedding, and groomingutensils, such as brushes and combs. The spray solution or suspension ofthe present invention may also be employed for the detection of ticks,mites, and fungi on the fur and skin of animals, such as dogs and cats.

[0019] Further provided by the present invention is a kit comprising asuspension containing a dye capable of binding or conjugating to achitin-containing organism in an amount effective to bind or conjugateto a chitin-containing organism and emit fluorescence upon exposure tolight, and a source of light that emits a wavelength capable of excitingfluorescence from the chitin-bound or chitin-conjugated dye.

[0020] The source of light that emits a wavelength capable of excitingfluorescence from the chitin-bound or chitin-conjugated dye may be asource of ultraviolet or visible light. Preferably, the source of lightcontained in the kit is a source of long wave ultraviolet light or shortwave visible light. The dye contained in the solution or suspensionwhich is a part of the kit provided by the present invention iscalcofluor white, Uvitex 3B, Rylux BA, Rylux BSU or Blankophor.

[0021] The present invention is described in the following ExperimentalDetails Section, which is set forth to aid in an understanding of theinvention, and should not be construed to limit in any way the inventionas defined in the claims which follow thereafter.

[0022] Experimental Details Section

[0023] Visualizing Nits

[0024] Human hair with attached P. capitis nits obtained as a wasteproduct after barbering were obtained and dipped into a 1% solution ofcalcofluor white for two minutes. The hair was removed and examined wetunder UV light. The nits were clearly seen as brightly fluorescent whiteovoid bodies against a non-fluorescent black background. Identificationwas easy, specific, and totally unambiguous.

[0025] Toxicity Data

[0026] The 1% solution was also spread on the dorsum of four hands ofvolunteers and allowed to remain continuously for 12 hours before beingwashed off. The solution caused no physical sensation and did notprovoke any physical reactions such as pruritus, erythema, induration ordiscoloration.

[0027] Visualizing Deer Ticks

[0028] Use as a visualizing agent for deer ticks was investigated aswell. Live deer ticks were gathered from a wooded area in WestchesterCounty, NY and sprayed with a 1% solution of Calcofluor white. The tickswere placed on a white cotton sheet and exposed to long range UV lightfrom a portable UV lamp. The ticks were easily visualized as brightlyfluorescent objects against a dark background.

[0029] In order to determine whether deer ticks and related arthropodsexhibit autofluorescence, several deer ticks were collected on a whitecotton sheet and subjected to exposure to long range ultraviolet light.There was no fluorescence demonstrated. The addition of a subsequentspray of Calcofluor white on the deer ticks with a follow up exposure toultraviolet light then showed intense fluorescence.

[0030] Commercial Applications

[0031] It is estimated that millions of children are treated andexamined for head lice each year. Untold millions of people a year areexposed to deer ticks and would be a likely group of people who wouldwant to screen themselves for deer tick bites.

[0032] In addition, countless pet dogs and cats are infected withringworm caused by Microsporum canis which also contains chitin.

[0033] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of the disclosure that various changesin form and detail can be made without departing from the true scope ofthe invention in the appended claims.

What is claimed is:
 1. A method for detecting a chitin-containingorganism on an area of a subject suspected of containing saidchitin-containing organism comprising the steps of: (a) contacting thesuspected area with a dye that is capable of binding to chitin andemitting fluorescence upon exposure to light; and (b) exposing thecontacted area with light to excite fluorescence of said dye and detectsaid chitin-containing organism.
 2. The method of claim 1 wherein thechitin-containing organism is an arthropod or a fungus.
 3. The method ofclaim 2 wherein the arthropod is a louse, nit, tick, or mite.
 4. Themethod of claim 2 wherein said louse or nit is Pediculus capitus,Pediculus humanus, Pediculus corporis or Phithirus pubis.
 5. The methodof claim 3 wherein the tick is Ixodes scapularis.
 6. The method of claim2 wherein the fungus is an Epidermophyton, Microsporum or Trichophytonspecies.
 7. The method of claim 1 wherein the dye is calcofluor white,Uvitex 3B, Rylux BA, Rylux BSU or Blankophor.
 8. The method of claim 1wherein the light source is ultraviolet light or visible light.
 9. Asuspension comprising a dye capable of binding to a chitin-containingorganism in an amount effective to bind to a chitin-containing organismand emit fluorescence upon exposure to light.
 10. The suspension ofclaim 9 wherein the dye is calcofluor white, Uvitex 3B, Rylux BA, RyluxBSU or Blankophor.
 11. The suspension of claim 9 wherein the dyecomprises 0.01-5% of the suspension.
 12. The suspension of claim 9 whichis a shampoo, lotion, cream, detergent or spray.
 13. A kit comprisingthe suspension of claim 9 and a source of light that emits a wavelengthcapable of exciting fluorescence from the chitin-conjugated dye.
 14. Asolution comprising a dye capable of binding to a chitin-containingorganism in an amount effective to bind to a chitin-containing organismand emit fluorescence upon exposure to light.
 15. The solution of claim14 wherein the dye is calcofluor white, Uvitex 3B, Rylux BA, Rylux BSUor Blankophor.
 16. The solution of claim 14 wherein the dye comprises0.01-5% of the suspension.
 17. The solution of claim 14 which is ashampoo, lotion, cream, detergent or spray.
 18. A kit comprising thesolution of claim 14 and a source of light that emits a wavelengthcapable of exciting fluorescence from the chitin-conjugated dye.